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p100: A Novel Proliferation-Associated Nuclear Protein Specifically
Restricted to Cell Cycle Phases S, G 2 , and M
By H.J. Heidebrecht, F. Buck, J. Steinmann, R. Sprenger, H.H. Wacker, and R. Parwaresch
By immunization with nuclear lysates of L428 cells, we
raised a monoclonal mouse antibody, Ki-S2 (IgG1 ). In Western blots, this antibody recognizes a nuclear antigen with
an apparent molecular mass of 100 kD, termed p100. Protein
sequencing of p100 showed that this is a hitherto unknown
protein. Immunohistochemical examination of cryostat and
paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the
nuclei of a fraction of proliferating cells. Cell sorting and
fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition
G1/S until the end of cytokinesis. During mitosis, this protein
is strictly associated with the spindle pole and with the mi-
totic spindle, whereas during S and G2 , p100 is diffusely
distributed throughout the cell nucleus. Immediately after
completion of cytokinesis, p100 was rapidly degraded. In
L428 cells, p100 is phosphorylated at least during mitosis. It
has a turnover time of about 1 hour. Studies on routinely
processed paraffin sections of specimens of malignant
lymphoma, benign and malignant nevocellular tumors, and
breast cancer showed that in all cases less than 40% of the
Ki-67–positive growth fraction expressed p100. Thus, p100
might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer
prognosis, beyond its general biologic relevance as a cell
cycle protein.
q 1997 by The American Society of Hematology.
R
pass the restriction point G1/S and contribute directly to the
magnitude of the proliferation compartment, or they may
senesce in G0 and succumb by apoptosis.15-17 In contrast to
G1 , the S, G2 , and M phases of the cell cycle are relatively
constant in duration.17 Moreover, cells in G1 make up the
largest fraction of the cycling subpopulation. Thus, G1 is the
major source of uncertainty in estimations of the growth
fraction.
We describe a hitherto unknown proliferation-associated
protein expressed exclusively from the transition G1/S until
the end of cytokinesis.
ECENT PROGRESS IN analyzing the structure and
function of cell cycle-associated proteins such as
cyclins, cyclin-dependent kinases, and their inhibitors has
considerably promoted our understanding of the mechanisms
controlling cellular proliferation.1-3 Among the large number
of proteins involved, only a few have proven useful for
monitoring proliferative activity and growth fraction. The
ability to identify antigens that are reliably associated with
cell proliferation in normal and neoplastic tissue, such as
Ki-67 and topoisomerase-IIa, by means of highly specific
antibodies has proven invaluable in diagnostic histopathology and cell biology.4-10
Both antigens have proven their diagnostic and prognostic
value in several retrospective and prospective studies.5,8
These proliferation markers have found widespread application, but some studies have failed to show a close correlation
between antigen expression and either the growth fraction
or the prognosis of tumors.11,12 The major shortcoming has
been an overestimation of the size of the proliferation compartment.11-13 One of the major reasons for this failure is the
fact that proteins such as Ki-67 and topoisomerase-IIa are
expressed in the cell cycle phases S, G2 , M, and G1.4,7,14
However, the G1 phase can easily be influenced by several
external and internal factors.15-17 Cells in G1 quiescence may
From the Department of Hematopathology and Lymph Node Registry, German Association of Pathologists at the University of Kiel,
Kiel, Germany; the Department of Cell Biochemistry and Clinical
Neurobiology, University of Hamburg, Hamburg, Germany; the Department of Immunology, University of Kiel, Kiel, Germany; and
the Division of Molecular Immunology, Forschungsinstitut Borstel,
Borstel, Germany.
Submitted December 9, 1996; accepted February 19, 1997.
Address reprint requests to Dr H.J. Heidebrecht, Department of
Hematopathology, University of Kiel, Michaelisstr. 11, D-24105
Kiel, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/9001-0020$3.00/0
MATERIALS AND METHODS
Generation of monoclonal antibodies (MoAbs). L428 cells (a
generous gift from Prof V. Diehl, Köln, Germany) were lysed and
the nuclei were washed and harvested by centrifugation. These nuclei
were used in incomplete Freund’s adjuvant to immunize female
BALB/C mice by three intraperitoneal injections at 10-day intervals.
After boostering, the splenocytes were fused with P3x63-Ag.8653
mouse myeloma cells. Hybridomas were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and subcloned.
Supernatants were screened for their reactivity with proliferating
cells in normal human tissue.
Tissue samples. The specificity of the MoAb was tested on approximately 4-mm–thick cryostat sections of nearly all normal human tissue types. In addition, Ki-S2 was tested on routinely processed paraffin sections of the same samples. Peripheral blood
mononuclear cells (PBMC) were prepared as cytospin slides. To
show the positive correlation between the MoAb Ki-S2 and other
established proliferation markers reported earlier from our laboratory,7,9 47 cases of non-Hodgkin’s lymphoma classified according
to the updated Kiel classification18 and the Working Formulation19
were studied using Ki-S2 and Ki-S5 on routinely processed paraffin
sections. The latter antibody is directed against the Ki-67 antigen.9
Twenty-six of the cases had been classified as chronic B-lymphocytic
leukemia (B-CLL) and 21 as Burkitt’s type high-grade B-cell
lymphoma. In addition, 47 cases of melanocytic lesions and 100
cases of stage I nodal negative ductal invasive carcinoma of the
breast were included.
Immunocytochemistry. Tissue samples fixed in 5% to 10% formaldehyde for an unknown period of time were routinely processed
for paraffin embedding. Approximately 4-mm–thick tissue sections
were fixed onto sialinized slides. For antigen retrieval, sections were
treated in a microwave oven as described earlier for other prolifera-
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226
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p100 PROLIFERATION-ASSOCIATED PROTEIN
227
tion-specific nuclear antigens.7,9 Cryostat sections and cytospin slides
were fixed in acetone at room temperature for 10 minutes and incubated with the primary antibody (Ki-S2 and Ki-S5 [specific for the
Ki-67 antigen]) for 30 minutes. The immunoreaction was visualized
by means of the alkaline phosphatase antialkaline phosphatase
(APAAP) method.20 The slides were briefly counterstained with
Meyer’s hemalum (all reagents from Dianova, Hamburg, Germany).
Immunofluorescence stainings of the L428 cells were performed
with the MoAb Ki-S2 and a goat antimouse polyclonal antibody
coupled with Cy3. A polyclonal rabbit anti Ki-67 serum (kindly
provided by Prof Dr J. Gerdes, Borstel, Germany) was used for
double immunofluorescence stainings.
Flow cytometry. PBMC from healthy donors were obtained by
density centrifugation of heparinized blood.21 PBMC (106/mL) were
cultured with 1 mg/mL phytohemagglutinin A (PHA; Wellcome,
Maidstone, UK) in RPMI 1640 supplemented with 10% human serum. Stimulated PBMC were harvested daily. Some of them were
used to prepare cytospin slides, which were immunostained with KiS2 and Ki-S5. Differential counts based on 500 cells each in Ki-S5
and Ki-S2 immunostained cytospin slides were correlated. Further
portions were fixed for 40 minutes with fresh paraformaldehyde
solution (0.5 g/mL) in phosphate-buffered saline (PBS) at 47C. The
cells were washed and subsequently permeabilized with 0.1% TritonX 100 in PBS. PBMC (5 1 105) were incubated with Ki-S2 at 47C
for 1 hour, washed, and labeled with fluorescein isothiocyanate goat
antimouse serum for 30 minutes. An anti–IL-2 antibody of the same
isotype (IgG1) was used as the negative control. Cytometry was
performed on a FACScan flow cytometer (BectonDickinson, Mountain View, CA). DNA staining was recorded on a linear scale and
antibody staining on a log scale. A total of 1,024 channels were
analyzed.
Cell sorter analysis. On day 3, PHA-stimulated (10 mL/mL)
PBMC were sorted twice with a cell sorter according to their DNA/
RNA (Hoechst 33342, 2 mg/mL; pyronin Y, 5 mmol/L) content into
G0/G1 and S/G2/M phase cells.22 Cytospin preparations of the two
sorted fractions were immunostained with Ki-S2 and Ki-S5 using
the APAAP technique.
Biolabeling and determination of the molecular mass of the antigen. L428 cells (3 1 106/mL) were labeled with [35S]methionine
(50 mCi) overnight. All further steps were performed on ice or at
47C. After intensive washing with ice-cold PBS, the cells were lysed
with 2% Triton-X 100, 1 mmol EDTA, and 1 mmol phenylmethanesufonyl fluoride (PMSF) in PBS, pH 7.4, for 5 minutes. After a
short centrifugation step, the pelleted nuclei were lysed with 0.35
mol/L NaCl, 1 mmol PMSF for 30 minutes. The lysate of the nuclei
was then centrifuged for 10 minutes at 15,000g, and the supernatant
was used for immunoprecipitation with Ki-S2. The MoAb Ki-S2
was coupled to rabbit antimouse IgG-coupled protein A Sepharose
CL-4B (Sigma, München, Germany). After 1 hour of incubation,
the immunoprecipitates were washed several times with a low (PBS,
0.4% Triton-X 100) and high salt solution (PBS, 0.5 mol/L NaCl,
0.4% Triton-X 100). After washing, the immunoprecipitate was
boiled in loading buffer under reducing conditions for 5 minutes.
The immunoprecipitate was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) at a gradient of 5%
to 10% or 7.5% to 15%. The gels were subjected to autoradiography
as described previously.23 A molecular weight standard (SDS-6H;
Sigma) served as reference.
To determine the turnover time of the Ki-S2 antigen, we labeled
L428 cells for 1 hour. Subsequently, the cells were intensively
washed and immunoprecipitation experiments were performed as
described above at various time intervals after the end of labeling.
In addition to immunoprecipitation, Western blotting was performed
with Ki-S2. In these experiments lysates of L428 were separated by
SDS-PAGE at a gel gradient of 5% to 10% or 7.5% to 15%) and
then transferred to nitrocellulose membranes overnight. After
blocking with 3% bovine serum albumin, the membranes were incubated with supernatant containing Ki-S2. Visualization was performed with 4-chloro 1-naphthol after the membranes had been incubated with peroxidase-conjugated rabbit antimouse IgG (Sigma).
To see whether the Ki-S2 antigen is subject to phosphorylation,
radiolabeling experiments were performed with [32P]orthophosphate
(Amersham Buchler, Braunschweig, Germany). L428 cells (1 1
107) were washed with phosphate-free buffer and then incubated in
Fig 1. Immunohistochemical
staining of a microwave-processed paraffin section of a formalin-fixed human tonsil with
Ki-S2. Cell nuclei in the dark zone
of a germinal center, which harbors proliferating cells, are
strongly positive. APAAP staining.
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228
HEIDEBRECHT ET AL
Table 1. PHA Stimulation of Peripheral Blood Lymphocytes
Days
n
Ki-S2
Ki-S3
% Ki-S2
0
1
2
3
4
5
6
8
8
8
8
8
8
8
ú1%
2%
19% { 5%
25% { 4%
17% { 3%
10% { 4%
9% { 2%
ú1%
5% { 2%
37% { 5%
64% { 9%
43% { 10%
34% { 9%
26% { 6%
40%
51%
39%
39%
29%
35%
Evaluation of immunohistochemical staining with Ki-S2 and Ki-S5.
Numbers represent mean and standard deviation of percentages.
Abbreviation: n, number of blood donors.
phosphate-free medium (minimal essential medium [MEM]; Bioconcept, Umkirch, Germany) supplemented with 20 mmol/L glutamine and 10% FCS for 30 minutes. Then, 1 mCi [32P]orthophosphate
(carrier-free) was added. After 1 hour, cells were harvested and
prepared for immunoprecipitation as described above.
Cell culture and synchronization. All experiments were performed with the cell line L428. L428 cells were grown in RPMI
1640 supplemented with 10% FCS and 2 mmol/L L-glutamine. Exponentially growing cells were arrested with 0.15 mg/mL colcemid
(Boehringer Mannheim, Mannheim, Germany) overnight.24
Protein sequencing. For protein sequencing experiments, a nuclear lysate preparation of 1 1 1010 HeLa cells was used. Immunoprecipitation was performed with Ki-S2 coupled to protein A Sepharose Cl-4B as described above. The immunoprecipitates were
separated by SDS-PAGE and then transferred by blotting to polyvinylidene fluoride (PVDF) membranes (Immobilon P; Millipore,
Eschborn, Germany). The membranes were stained with Coomassie
brilliant blue R 250. After destaining with 50% methanol, the protein
band was excised from the membrane and further processed for
sequencing. Because attempts to sequence the intact proteins from
the blot membrane were unsuccessful, the protein was digested with
Lys C, as described by Bauw et al,25 and the proteolytic fragments
were separated by narrowbore high-performance liquid chromatography (130 A; Applied Biosystems, Weiterstadt, Germany) on a
reverse-phase column (Vydac C4; 300 A pore size; 5 mm particle
size; 2.1 1 125 mm). Peptides were eluted with a linear mobile
phase gradient (0% to 80% B in 50 minutes; solvent A, water/0.1%
TFA; solvent B, 70% acetonitrile/0.09% trifluoroacetic acid [TFA])
at a flow rate of 200 mL/min. Peptide-containing fractions detected
at 214 nm were collected manually into siliconized Eppendorf tubes
and frozen immediately. Protein sequences of 10 peptides were determined by standard Edman degradation on an automatic sequencer
(473 A; Applied Biosystems).
Table 2. Immunohistochemical Staining of Different Tumor Types:
Comparison of Ki-S2 and the Anti–Ki-67
Antigen-Specific MoAb Ki-S5
Diagnosis
n
Ki-S2
B-CLL
Burkitt’s lymphoma
Breast cancer
Malignant melanoma
Benign nevi
26
21
100
18
29
4% { 3%
95% { 18%
17% { 6%
16% { 6%
ú1%
Ki-S5
14%
36%
43%
48%
4%
{
{
{
{
{
6%
9%
18%
22%
3%
% Ki-S2
28%
37%
39%
33%
25%
Numbers represent the mean and standard deviation of percentages. All tissue samples were routinely processed paraffin sections.
Abbreviation: n, number of cases.
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Fig 2. Flow cytometric analysis of unstimulated (day 0) and PHAstimulated PBMC (days 1 through 3). DNA staining was registered
on a linear scale (FL1-A) and Ki-S2 staining on a log scale (FL2-H).
Less than 1% of the unstimulated PBMC were positive. Twenty-four
hours after PHA stimulation, a small population of the G1 cells became positive. On days 2 and 3, all cells in S/G2 and M phases bound
Ki-S2.
RESULTS
Immunoreactivity of Ki-S2 in normal tissue. Ki-S2 was
shown to be a mouse monoclonal IgG1 antibody that specifically detects an antigen in the nuclei of proliferating cells
in sections of frozen and paraffin-embedded tissues. There
was no difference in the number of positive cells and in the
intensity of the reaction between frozen sections and paraffin-embedded samples. The distribution of this nuclear antigen in normal human tissue was found to be restricted to
sites harboring cycling cells, such as the germinal centers
of lymphoid tissue (Fig 1). Nonspecific cross-reactivities in
normal human tissue were not observed. Extensive immunohistochemical investigation of all normal tissue types showed
that p100 and the Ki-S2 antibody are highly specific to the
nuclei of cycling cells in characteristic tissue sites. In the
epithelial coverings, a few basal epithelial cells and a moder-
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p100 PROLIFERATION-ASSOCIATED PROTEIN
229
Fig 3. Staining of L428 cells with Ki-S2 and a Cy3labeled goat antimouse polyclonal antibody. During
the different phases of mitosis, the Ki-S2 antigen is
strongly associated with the spindle poles and the
mitotic spindle, whereas in the S and G2 phases, cells
the antigen is diffusely distributed throughout the
cell nucleus.
ate number of suprabasal epithelial cells showed a positive
reaction. In the mucosa of the gastrointestinal tract, Ki-S2–
positive cells were restricted to the lower part of the crypts.
In lymphoid tissue, the dark zone of the germinal centers of
lymphoid B follicles was highly reactive, whereas the number of positive cells did not exceed 5% in other areas of
the lymph nodes. In testicular tissue, spermatogonia reacted
strongly, whereas mature spermatozoa were invariably negative. In the bone marrow, promyelocytes, myelocytes, proerythroblasts, and erythroblasts (E1 and E2) were positive,
whereas mature granulocytic cells and erythroblasts were
consistently negative. Normal nonstimulated PBMC were
negative for Ki-S2 and Ki-S5, and only occasional cells
(õ1%) showed faint nuclear reactivity. After stimulation
of PBMC with PHA, the expression of the Ki-S2 antigen
increased, paralleling that of the Ki-S5–reactive Ki-67 antigen. On day 3, 25% { 4% of the stimulated cells expressed
the Ki-S2 antigen. The Ki-S2 antigen expression correlated
positively with the expression of the Ki-67 antigen determined by the MoAb Ki-S5 (r Å .89, P õ .01; Table 1).
Expression of Ki-S2 in neoplasms. With Ki-S5, the Ki67 proliferation marker included in this study, the cases of
B-CLL showed a low percentage of positive cells. In contrast
to this typical low-grade B-cell lymphoma, considerably
higher values were detected in the Burkitt’s lymphomas. In
all lymphoma cases there was a significant (P õ .01) positive
correlation (r Å .97) between the results achieved with the
antibody recognizing the Ki-67 antigen (Ki-S5) and with KiS2 (Table 2).
Similar results were obtained when other neoplasms were
studied with Ki-S2 in comparison to Ki-S5. Cases of stage I,
nodal-negative ductal invasive adenocarcinoma of the breast
showed 42% { 11% Ki-67–positive tumor cells, as detected
with the antibody Ki-S5. Only around 40% of the Ki-67–
positive cells (17% { 9%) expressed the Ki-S2–reactive
nuclear antigen (Table 2).
Of the melanocytic tumors, in benign nevi (n Å 29), less
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than 2% of the cells were positive for the Ki-67 antigen. KiS2 was also detectable in less than 1%. In cases of malignant
melanoma (n Å 18), 38% { 12% of the cells showed Ki-67
expression, whereas only 15% { 6% of the cells were positive for Ki-S2. The quantitative results with both antibodies
showed a highly significant correlation (r Å .97 and .98; P õ
.01) for all tumor types (Table 2). All immunohistochemical
stainings to determine the expression of the Ki-S2 antigen
in the different neoplasms were performed on routinely processed paraffin sections.
Cell sorting and flow cytometry. Cell sorting of stimulated PBMC into highly purified G0/G1 and S/G2/M cell populations showed a clear difference with respect to Ki-S2
expression. Whereas only occasional (ú1%) positive cells
were detectable in the G0/G1 fraction, slides prepared from
the S/G2/M phase fraction showed considerably higher reactivity, exceeding 95% of the sorted cells. These results were
well in line with those obtained by fluorescence-activated
cell sorting (FACS) analysis (Fig 2). Twenty-four hours after
stimulation, the percentage of positive cells was low. It increased on days 2 and 3, as the cells traversed the S, G2 ,
and M phases of the cell cycle.
Immunofluorescence staining showed that the antigen recognized by the antibody was diffusely distributed throughout
the cell nuclei during S and G2 phases. In M phase, the
antigen was strictly associated with the spindle poles and
the mitotic spindles (Fig 3). Double immunofluorescence
stainings with the MoAb Ki-S2 and a polyclonal anti–Ki-67
antigen serum showed that Ki-S2 antigen expression ceased
immediately after the termination of cytokinesis (data not
shown).
Determination of the molecular mass and first characterizations of the Ki-S2 antigen. The molecular mass of the
Ki-S2 antigen in L428 cells was determined by immunoprecipitation and Western blotting. Under reducing conditions,
one antigen with a molecular mass of about 100 kD, termed
p100, could be detected with both methods (Fig 4). An anti-
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230
HEIDEBRECHT ET AL
mologies with already characterized proteins, amino acid
sequences were analyzed. For this purpose, nuclear lysates
of 1 1 1010 HeLa cells were used for immunoprecipitation.
The precipitated antigen was blotted onto an Immobilon P
membrane and stained with Coomassie blue (Fig 7). This
antigen preparation was monitored by Western blot experiments with the MoAb Ki-S2. Only antigen preparations with
a molecular mass of about 100 kD that could be stained with
the MoAb Ki-S2 were used for protein sequencing. Because
attempts to sequence the N-terminus of the Ki-S2 antigen
p100 failed, the protein was digested with Lys C. Ten peptides of two independent immunoprecipitations were sequenced. The sequences of these peptides were compared
with protein sequences from various protein data banks (PIR,
Swiss Prot, Geneva, Switzerland). No homology with known
proteins could be detected.
Fig 4. Western blot of lysed L428 cells using the MoAb Ki-S2 (B)
after SDS-PAGE (gradient gel 5% to 10%). Ki-S2 detects a protein of
about 100 kD. (A) Control experiment with an MoAb of the same
isotype (IgG1) specific for topoisomerase-IIa (170 kD). (C) Control
without primary antibody. Molecular weight standards are shown
on the right (in kilodaltons).
body of the same isotype (IgG1) specific for topoisomeraseIIa (170 kD) served as positive control.26 After L428 cells
were radiolabeled with [32P]orthophosphate, a signal could
be detected that was strongly enhanced when L428 cells
were arrested in mitosis with colcemid for 18 hours (Fig 5).
After 18 hours, about 35% of the L428 cells were in mitosis.
To determine the turnover time of the Ki-S2 antigen, L428
cells were labeled with [35S]methionine for 1 hour. The cells
were washed and immunoprecipitation experiments were
performed at different time intervals after the end of labeling.
Two hours after the end of labeling only a weak signal could
be detected, which indicates a short half-life of about 1 hour
for p100 (Fig 6).
Protein sequence data. To look for protein sequence ho-
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Fig 5. Immunoprecipitation experiment with Ki-S2 on L428 cells
after the cells were labeled with [32P]orthophosphate (A). In lane B,
L428 cells were arrested with colcemid (0.15 mg/mL) overnight before
labeling with [32P]. (C) Isotype control experiment. Molecular weight
standards are shown on the right (in kilodaltons).
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p100 PROLIFERATION-ASSOCIATED PROTEIN
231
DISCUSSION
We report a new formalin- and paraffin-resistant, proliferation-associated nuclear antigen with a molecular mass of
about 100 kD. This antigen, p100, is specifically recognized
by a mouse IgG1 MoAb designated Ki-S2. Extensive immunohistochemical investigations on frozen sections and paraffin-embedded samples of all normal tissue types showed that
p100 and the Ki-S2 antibody are highly specific for the
nuclei of cycling cells in characteristic tissue sites. In the
epithelial coverings, a few basal epithelial cells and a moderate number of suprabasal epithelial cells showed a positive
reaction. In the mucosa of the gastrointestinal tract, Ki-S2–
positive cells were restricted to the lower part of the crypts.
In lymphoid tissue, the dark zone of the germinal centers of
lymphoid B follicles was highly reactive, whereas the number of positive cells did not exceed 5% in other areas of the
lymph nodes. In testicular tissue, spermatogonia showed
high reactivity, whereas mature spermatozoa were invariably
Fig 7. A Coomassie blue staining of a PVDF membrane after an
immunoprecipitation experiment using cell nuclei of 1 Ì 1010 HeLa
cells with Ki-S2. The 100-kD protein band of this experiment was
excised and used for digestion with Lys C and peptide sequencing.
Molecular weight standards are shown on the right (in kilodaltons).
Fig 6. Determination of the turnover time of the Ki-S2 antigen.
L428 cells were labeled for 1 hour with [35S]methionine and intensively washed. Immunoprecipitation experiments were performed
immediately after the end of labeling (A) and 90 minutes (B), 120
minutes (C), and 180 minutes (D) after the end of labeling. (E) Isotype
control experiment after the end of labeling. Molecular weight standards are shown on the right (in kilodaltons).
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negative. In the bone marrow, promyelocytes, myelocytes,
proerythroblasts, and erythroblasts (E1 and E2) were positive, whereas mature granulocytic cells and erythroblasts
were consistently negative. Although nearly all normal leukocytes of the peripheral blood were negative for this antigen, PHA stimulation induced increasing expression of KiS2 from less than 1% on day 0 to a maximum of 25% {
4% on day 3.
Ki-S2 showed the same distribution pattern in normal tissue as the established proliferation markers Ki-67 and topoisomerase-IIa.4,7,9,26 However, in quantitative terms, Ki-S2
immunolabeled only 30% to 40% of the Ki-67– and topoisomerase-IIa –expressing cell population.27 All computations showed a significant positive correlation between the
expression rates of the two antigens.
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HEIDEBRECHT ET AL
In neoplastic tissue, these antigens showed similar behavior. In cases of indolent low-grade B-cell lymphomas, such
as B-CLL, and benign tumors, such as benign nevi, the expression of the p100 and Ki-67 antigens was extremely low.
High activity levels were found for aggressive B-cell
lymphomas of Burkitt’s type and for other malignant tumors
such as ductal invasive cases of breast carcinomas, whereby
the p100 level did not exceed about 40% of the Ki-67 levels,
on average. The highest Ki-67 antigen expression, exceeding
90% of the tumor cells, was found in cases of Burkitt’s
lymphoma. p100 expression was also high, although considerably lower than Ki-67 expression. These results imply that
p100 is expressed in only a certain portion of the cell cycle
phases.
Cell sorting studies, separating cells in G0/G1 from cells
in the S, G2 , and M phases of the cell cycle, showed that,
unlike Ki-67, the p100 antigen was selectively expressed
during the cell cycle phases S, G2 , and M. Flow cytometric
studies confirmed these results. In addition, these results lend
support to conclusions deduced from immunoprecipitation
studies on [35S]methionine-labeled cells at different time intervals, which implied that p100 is rapidly degraded after
the end of cytokinesis and promptly expressed as the cells
traverse the G1/S restriction point. The turnover time of p100,
estimated from the degradation kinetics, is around 1 hour.
Immunofluorescence studies with Ki-S2 or Ki-S2 in combination with a polyclonal anti–Ki-67 antibody in double immunofluorescence stainings show that, until mitosis, p100 is
evenly distributed throughout the nucleus, whereas during
mitosis, it is strictly associated with the spindle poles and
the mitotic spindle.
Immunoprecipitation of radiolabeled p100 showed a molecular mass of about 100 kD. This result was also confirmed
by Western blotting. Amino acid analysis of the purified
protein after proteolytic degradation showed no significant
homology with any protein sequence known so far. Thus,
p100 is a new nuclear proliferation-associated protein selectively expressed in the S, G2 , and M phases of the cell cycle.
p100 differs in the above-mentioned features from all other
known cell cycle-associated proteins and from those associated with proliferating cells, such as p350/320 (Ki-67 antigen), p170 (topoisomerase-IIa), p36 PCNA (proliferating
cell nuclear antigen), p210 (nuclear matrix antigen), p125
(mitotin).27-31 Nuclear proteins with a nearly identical molecular mass of 100 kD, such as nucleolin and inner centromere
binding proteins (INCENPs), have no sequence homologies
with the 10 peptides resolved from p100 by proteolytic digestion. In line with these considerations, the corresponding
immunohistochemical studies show a completely different
staining pattern.32,33
No information is available on the functional aspects of
p100. Its restricted expression between the G1/S restriction
point and termination of cytokinesis, as detected by cell
sorter analysis and by double immunofluorescence stainings,
and its close association with the spindle poles, plus the
fact that p100 is phosphorylated during the M phase, might
indicate a role in the mechanics of cytokinesis. Activation
by phosphorylation is a phenomenon regularly found in cell
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cycle-associated proteins, such as PCNA and topoisomeraseIIa.34-36
To our knowledge, p100 is the first cell cycle-associated
protein with such a restricted expression pattern. The highly
specific MoAb Ki-S2 can probably replace S phase labeling
with radiolabeled DNA precursors37 and thus yield proliferation data with a closer correlation to tumor prognosis.
Considering the importance of determining cell proliferation in cell biology and clinical oncology,11,38,39 several different methods for the assessment of cellular proliferation
have been developed. One of the most effective ways to
survey the proliferative activity of a cell population is the
mean population doubling time.38 However, this is feasible
only for tissue culture conditions. This also applies to radiomonitoring or immunohistochemical detection of DNA precursors, such as [3H]thymidine and bromodeoxyuridine
(BrDU). Like Ki-S2, antibodies to BrDU are expressed in
about 30% of the Ki-67–positive cells.37 The introduction
of immunohistochemical methods for monitoring cellular
proliferation in cell suspensions or fixed tissue sections using
MoAbs to proliferation-specific cell proteins, such as Ki-67
antigen and topoisomerase-IIa, has been of considerable
help in cell biology and in the evaluation of tumor
growth.7,9,40 These antigens are also expressed during the G1
cell cycle phase. Because the G1 phase, which accounts for
a major part of cell cycle, varies extremely in duration, such
markers are unreliable for tumor entities with prolonged G1.
The immunohistochemical assessment of p100 should become a powerful tool for diagnostic and prognostic purposes.
Further studies on this hitherto unknown proliferation-associated protein will increase our knowledge of the cell cycle
puzzle.
ACKNOWLEDGMENT
The authors thank M. Hauberg and G. Jopp for their excellent
technical assistance, K. Herwartz for performing the flow cytometry
experiments, and K. Dege for helping prepare the manuscript.
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1997 90: 226-233
p100: A Novel Proliferation-Associated Nuclear Protein Specifically
Restricted to Cell Cycle Phases S, G 2 , and M
H.J. Heidebrecht, F. Buck, J. Steinmann, R. Sprenger, H.H. Wacker and R. Parwaresch
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